Storage

It is wrongly assumed that Liquid Nitrogen (LN2) is sterile and, in fact, when manufactured, it usually has a very low particulate, water, and microbial content. However, it has been demonstrated that contamination of samples may occur during the storage of cells in LN2. The two main potential sources of contamination are other cryopreserved samples stored in the same vessel and the LN2 itself.

In 2003, Bielanski found that virtually all cryotanks tested, that had been in continuous service for 35 years, had bacterial and fungal contamination in the LN2 detritus. The most prevalent bacterial strain detected was Stenotrophomonas maltophilia, that has been shown to affect sperm motility and severely suppress embryonic development.Over the storage time, due to the exposure of cryotanks to the laboratory environment during refilling and handling of specimens, ice crystals will form on the walls of the vessels. Aggregated ice and sediment may entrap viruses, bacteria and fungal spores, posing a risk of microbial transmission to stored samples.

In 2004, Morris analyzed the ice sediments that accumulate in dewars during long-term storage. Bacteria were identified in all samples and filamentous fungi in 9 out of 10 samples. Morris concluded that the micro-organisms derived from the general laboratory environment and not from IVF samples stored in the liquid nitrogen and that the large variation in the microbial content between different samples of ice in the same dewar was indicative of contamination having occurred on many independent occasions over a number of years.

For this reason, all cryotanks used for the storage of biological samples should be considered potentially contaminated with at least environmental microorganisms.

Most microorganisms can survive storage at low temperatures, including in LN2 (-196° C). Furthermore, many ingredients of embryo culture media and semen extenders may act as stabilizers for microorganisms at freezing temperatures. Germplasm is usually stored in large capacity cryotanks, some of which may accommodate hundreds of thousands of straws or other sample containers. This may create a potential cross-contamination of clean samples in case of breaking or leaking of infected samples into the LN2.

For this reason, Bielanski in his latest review of the status of the science, recommended that cryotanks should be periodically decontaminated using an efficient disinfectant to decrease the risk of cross-contamination. This procedure is cumbersome and time consuming. NTERILIZER now offers a method to decontaminate cryotanks efficiently and effectively using UV radiation.